Efficient vaccine production requires the growth of large quantities of virus produced in high yields from a host system. Different types of virus require different growth conditions in order to obtain acceptable yields. The host in which the virus is grown is therefore of great significance. As a function of the virus type, a virus may be grown in primary tissue culture cells, established cell lines or in embryonated eggs, such as those from chickens.
Some of the mammalian cell lines which are used as viral host systems produce virus at high yields, but the tumorigenic nature of such cells invokes regulatory constraints against their use for vaccine production. In fact, the applicable guidelines of the World Health Organization (WHO) indicate that only a few cell lines are allowed for virus vaccine production.
There are three general types of influenza viruses, Type A, Type B and Type C, which are defined by the absence of serological crossreactivity between their internal proteins. Influenza Type A viruses are further classified into sub-types based on antigenic differences of their glycoproteins, the hemagglutinin (HA) and neuraminidase (NA) proteins. Humans are susceptible to diseases caused by infection with each of influenza Type A, B, and C viruses.
Currently, the most significant causes of influenza infections in humans are those attributable to Type B and to subtypes H1N1 and H3N2 of influenza type A. Accordingly, antigens of Type B and of subtypes H1N1 and H3N2 of influenza Type A are those which are generally incorporated into present seasonal influenza vaccines. The vaccines currently available have protection rates ranging from 75-90%. However, influenza strains that may cause pandemic infection are, for instance, of the H5N1 subtype which are not protected against by typical influenza vaccines. H2, H7 and H9 subtypes also have pandemic potential. See for example Koopmans et al., Lancet 363:587-93, 2004; Joseph et al., Md Med. 6:30-2, 2005; Cameron et al., virology 278:36-41, 2000; and Huber et al., Pediatri Infect Dis 27(10 Suppl):S113-17, 2008.
Live attenuated influenza virus vaccines have recently been approved for use in the United States. Many current vaccines are for seasonal influenza infection and contain two components of influenza A, H1N1 and H3N2, and an influenza B component. Over the last several years, at least one of the components was changed each year due to antigenic drift and mutation in the influenza proteins. Clinical isolates of human influenza virus are taken from infected patients and are reassorted in embryonated chicken eggs with a laboratory-adapted master strain of high-growth donor virus, the A/PuertoRico/8/34 (H1N1) influenza strain (U.S. Pat. No. 7,037,707). The goal of the reassortment is to increase the yield of candidate vaccine strains achieved by recombining at least the HA or NA genes from the primary clinical isolates, with the six internal genes of the master strain donor virus. This strategy provides high growth reassortants having antigenic determinants similar to those of the clinical isolates (Wood, J. M. and Williams, M. S., Textbook of Influenza. Blackwell Science Ltd, Oxford, 1998; Robertson et al, Biologicals, 20:213, 1992). The vaccines are prepared by growing this reassorted viral strain in embryonated eggs and then inactivating the purified virus by chemical means.
Highly pathogenic avian influenza viruses are capable of causing severe respiratory disease and mortality in birds. This feature is known only for HA of H5 and H7 subtypes. Because of the ability of some avian viruses to transmit to humans, they also become a human health concern. The pathogenicity of avian influenza viruses is a polygenic property but the HA protein has been shown to have the primary role in infection. A prerequisite for virus infectivity is the cleavage of the precursor HA protein (HAO) to HA1 and HA2 subunits. This cleavage releases the peptide that is responsible for the fusion of viral and endosomal membranes. Low pathogenic viruses express surface HA molecules that are activated only by trypsin-like proteases secreted by respiratory or gastrointestinal cells (Perdue et al., Virus Res 49:173-86, 1997). The cleavage site of highly pathogenic viruses is changed in a way that it can be cleaved by different cell proteases, especially by the ubiquitous furin that is present in the majority of cells (Steinhauer D., Virology 258:1-20, 1999; Swayne D., Vet Pathol 34, 557-67, 1997). All highly pathogenic strains invariably contain multiple basic residues (like R and K) at the cleavage site (Perdue et al., supra). Therefore, the virus can rapidly spread and replicate in nearly any organ and induce a systemic infection.
Attenuated H5N1 reassortant viruses have been produced by reverse genetics (R G, Palese et al., Proc. Natl. Acad. Sci. (USA) 93:11354-58, 1996), using the HA and NA genes from an H5N1 strain and inserting these genes into a “backbone” virus comprising the remainder of the internal influenza viral genes from a virus that is less pathogenic. The backbone often used is derived from the prototype strain A/Puerto Rico/8/34 (A/PR/8/34) of subtype H1N1 that is highly adapted to growth in eggs, and only the two surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are derived from an H5N1 virus. These reassortant viruses are designed for vaccine production in eggs, but the growth of the existing reverse genetic reassortants known in the art is poor in eggs and in cell culture. For example, Suguitan et al. (PLoS Medicine 3:1541-54, 2006) describe use of the cold-adapted Ann Arbor (ca AA) backbone to generate viruses having the HA and NA genes from H5N1 strains, A/Hong Kong/213 and A/Vietnam/1203/2004. These viruses demonstrated attenuation in vivo, i.e, were not lethal in chickens, and also exhibited some protective effects in mice rechallenged with wild type H5N1 virus. Shengqiang et al. (J Infectious Dis 179:1132-8, 1999) also produced recombinant virus using a pandemic Hong Kong strain HA and NA genes and Ann Arbor strain internal genes. Growth of these viruses in eggs, however, is less than optimal.
Likewise, Ming, et al., Chin. J. Biotech. (2006) 22:720-726, described, inter alia, a reassortant virus comprising internal genes from H9N2 viral strains, with a modified HA gene from an H5N1 strain and an NA gene from H2N3. Shi, et al., Vaccine (2007) 25:7379-7384, described, inter alia, a reassortant virus having internal genes from an H9N2 strain, with a modified HA and a wild-type NA gene from an H5N1 strain. Hickman, et al., (J. Gen. Virol. 89:2682-2690, 2008), described, inter alia, a vaccine comprising backbone genes from an H9N2 strain rescued with HA and/or NA genes from H1N1, H5N1 and H7N2 strains.
Recent studies have attempted to generate vaccines that have been grown in mammalian cell culture to avoid the problems commonly observed with growth of virus in egg, e.g., cost of upkeep and maintenance of egg facilities, purification of virus from egg protein, and allergy to residual egg protein. U.S. Pat. Nos. 6,146,873 and 7,132,271, among others, discuss development of cell culture lines that are capable of producing vaccine quality virus. See also Kistner et al., (Vaccine 16:960-8, 1998), which discloses VERO cells adapted for improved viral growth and vaccine production are described, and Ehrlich et al, (New Eng J Med 35:2573-84, 2008), which described, inter alia, a formalin-inactivated H5N1 whole virus vaccine grown in Vero cells.
Thus, there remains a need in the art to produce a pandemic or seasonal influenza vaccine having increased antigenicity compared to previous vaccines, that can elicit a good immune response in a host without causing infection, and which exhibits robust growth in mammalian cell culture.